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1997 Oxford University Press Human Molecular Genetics, 1997, Vol. 6, No. 10 Review 1613–1624 Homologous pigmentation mutations in human, mouse and other model organisms Ian J. Jackson MRC Human Genetics Unit, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, UK Received May 13, 1997 Mouse coat colour genes have long been studied as a paradigm for genetic interactions in development. A number of these genes have been cloned and most correspond to human genetic disease loci. The proteins encoded by these genes include transcription factors, receptor tyrosine kinases and growth factors, G-protein coupled receptors and their ligands, membrane proteins, structural proteins and enzymes. Many of the mutations have pleiotropic effects, indicating that these proteins play a wider role in developmental or cellular processes. In this review I tabulate the available data on all pigmentation genes cloned from mouse or human, and I focus on three particular systems. One family of genes, including LYST and HPS/ep, shows the relationship between melanosomes and lysosomes. The G-protein coupled receptor, endothelin receptor-B, and its ligand, endothelin-3, are required for the development of both melanocytes and enteric neurons. The melanocortin-1 receptor is expressed only on melanocytes, but mutations that cause overexpression of agouti protein, an antagonist of the receptor, result in obesity, and highlight a role of melanocortins in weight homoeostasis. INTRODUCTION Mouse coat colour genes were among the first mammalian mutant genes known. For most of this century they have been studied as a model of the way genes interact to regulate the developmental and cellular function of the pigment cell or melanocyte. There are about 80 classical mutations that have an effect on mouse coat colour. To date, 17 of the genes underlying these mutations have been cloned, and in 14 cases there is a human mutant phenotype described for the corresponding human gene. There are good molecular data too that at least six have mutations in other mammalian species. In contrast there is only a single human genetic pigmentation disease locus that does not correspond to a known mouse mutation. This information is summarised in Table 1 which also provides references for all the loci. Much of the molecular information has also been summarised in recent reviews (1,2). In this article I will concentrate on the molecular genetics of three systems in which there has been significant recent progress in mouse, human and other mammals. Each of these systems highlights the fact that developmental and cellular processes that characterise melanocytes may also be found in other cell types. Mutations affecting melanocytes, which are readily found because of their effect on pigmentation, will quite often be found to affect other processes. (Nomenclature note: in this review, gene symbols are italicised, their protein products are non-italic. Human gene and protein symbols are all capitalised. Where a mouse gene product is known, the gene and protein symbols are lower case with an initial capital.) I will first summarise work on two genes that encode proteins involved in the structure and/or function of both melanosomes and lysosomes and which are members of a larger family of functionally related genes. Secondly, two G-protein coupled receptors have been identified that are necessary for normal melanocyte development and function. These are the endothelin receptor B (Ednrb/EDNR3) and the melanocortin 1 receptor (Mc1r/MC1R). Coat colour mutations in mouse Ednrb and its ligand endothelin 3 (Edn3) have been known for many years, but their molecular identity only became known recently. Similarly, mutations in a pair of genes that had opposing effects on the type of pigment made by hair follicle melanocytes had been studied for a long time before their identity was established as the melanocortin 1 receptor and a functional antagonist of this receptor, the agouti protein. Throughout pigmentation genetics the similarities and differences between phenotypes of mutations of the same gene in different species have proved very informative for deducing how the gene products function in normal development and physiology. MELANOSOMES AND LYSOSOMES The site of melanin synthesis within melanocytes is an organelle called the melanosome. This becomes filled with melanin before it is transferred from the cell to neighbouring keratinocytes. A large body of evidence suggests a close relationship between melanosomes and lysosomes. Several proteins, such as lysosomal-associated membrane protein-1 (LAMP-1), and at least five lysosomal hydrolases are found in fractionated melanosomes (3,4). Furthermore, if the melanogenic enzymes, tyrosinase and TRP-1, which normally localise to the melanosome, are expressed in non-melanogenic cells, they are found in lysosomes (5). Genetic evidence in support of this relationship comes from a series of a dozen mouse loci, mutations Tel: +44 131 467 8409; Fax: +44 131 332 2471; Email: [email protected] 1614 Human Molecular Genetics, 1997, Vol. 6, No. 10 Review of which have an effect on pigmentation because of abnormal melanosomes but also affect the secretion of kidney lysosomal enzymes and have defective platelet dense granules. This last defect gives rise to a deficiency of the platelet storage pool, and hence a prolonged bleeding time (6–9). At least two human disorders, Chediak–Higashi syndrome (CHS) and Hermansky– Pudlack syndrome (HPS), have a similar range of disorders and have now been shown to be homologous to two of the mouse mutations, beige (bg) and pale ear (ep). Griscelli disease is a disorder similar to CHS, affecting pigmentation and immune function. The gene has recently been shown to encode Myosin-V, which is mutated in the dilute mouse mutation (10). CHS and beige Although the storage platelet deficiency family of mutations have many similarities, there are also differences between them. It had long been suspected on the basis of similarity of phenotype that the mouse beige mutation and human CHS (OMIM 214500) are homologous. Additional evidence came from the demonstration that the lysosomal trafficking defect seen in fibroblasts from mutant mice and humans did not complement following cell fusion (11). Fibroblasts from a strain of mink, known as Aleutian, also failed to complement the CHS or beige defect, indicating that this too is a homologous mutation. Initial mouse crosses mapped beige close to a marker, Nid, on chromosome 13 (12). This segment of the mouse genome is homologous to the telomeric end of human chromosome 1q, and CHS was mapped to this region by linkage in families and by homozygosity in individuals from consanguinous families (13,14). The complementation assay in somatic cells was a key to isolating the mouse beige gene. Perou et al. (15) were able to use YACs containing Nid to complement the lysosomal defect in beige fibroblasts, and subsequently were able to isolate from the YAC 6.5 kb of cDNA from a gene within 100 kb of Nid which was mutated in certain beige alleles (Table 2). A second group produced a high resolution genetic map and a YAC contig around beige which they used to select cDNAs including ∼5 kb of sequence that was mutated in certain other beige alleles (16) (Table 2). Surprisingly, the two cDNAs did not overlap, but once the full length, 12 kb, cDNA from humans was isolated it became clear that the two cDNAs were from the 3′ and 5′ ends, respectively of the same mRNA (17,18). This mRNA encodes a large protein named LYST (lysosomal trafficking regulator) that consists of 3801 amino acids. Analysis of CHS patients has identified eight mutations to date in this gene (16–19) (Table 2), which all produce truncated products. Table 1. Summary of all cloned pigmentation mutations of mouse and human Class of encoded protein Gene product Mouse classical mutation Human disease mutation Transcription factor PAX3 splotch Waardenburg syndrome 1 Other species Ref. 24–26 Waardenburg syndrome 3 Receptor tyrosine kinase and ligand G-protein coupled MITF microphthalmia Waardenburg syndrome 2 KIT dominant white spotting (W) Piebaldism Mast cell factor (Kit-ligand) steel none described Endothelin receptor B piebald Hirschsprung disease, Endothelin 3 lethal spotting receptor and ligands 24,76,77 pig 78–82 rat see Table 4 83–85 Shah–Waardenburg disease Hirschsprung disease see Table 5 Shah–Waardenburg CCHS Melanosome and Melanocortin 1-receptor extension, sombre, associated with skin type I, horse, cattle, chicken, (MC1R) recessive yellow and/or red hair guinea-pig, fox Agouti protein non-agouti; none described fox (MC1R antagonist) dominant yellow mink see Fig. 1 57,65,67,68,86 LYST beige Chediak–Higashi syndrome HPS/Hps pale ear Hermansky–Pudlak syndrome Melanosome distribution Myosin V dilute Griscelli disease yeast? 10,87,88, Copper transport ATP7A mottled Menkes disease yeast? 89–93 Melanogenic enzymes Tyrosinase albino 1,2,94–97 lysosome function see Table 2 see Table 3 occipital horn syndrome Other membrane proteins Oculocutaneous albinism I rabbit Ocular albinism Medaka fish rat Tyrosinase Related Protein-1 brown OCA-III Dopachrome tautomerase slaty none described 1,98–101 102,103 no function ascribed, pink-eyed dilution OCA-II 2,97,104–108 silver none descibed 109,110 none ocular albinism I 111–114 possible transport function Melanosomal matrix protein, possibly enzyme no function ascribed 1615 Human Molecular Genetics, 1997,1994, Vol. Vol. 6, No. Review 1615 Nucleic Acids Research, 22,10 No. 1 Table 2. Mutations of the LYST (CHS/beige) gene Species Mutation Molecular defect mouse bg partial LINE1 insertion: abnormal splicing, frameshift, protein truncation 15 bg 8J CGA→TGA, nonsense mutation, protein truncation 15 bg 11J 5 kb deletion: predicts incorrect splice, protein truncation 16 bg 2J >5× decrease in mRNA: molecular nature not defined 16 childhood CHS homozygous 1 bp deletion, codon 489, frameshift, protein truncation 17 childhood CHS homozygous 1 bp insertion, codon 633/634, frameshift, protein truncation 18 childhood CHS homozygous 1 bp deletion, codon 3197, frameshift, protein truncation 18 childhood CHS homozygous CGA→TGA, nonsense mutation, codon 1029 19 childhood CHS compound heterozygote; 2 bp deletion, codon 1026, frameshift, protein truncation 19 human Ref. Second mutation not detected childhood CHS compound heterozygote, 1 bp insertion codon 40, frameshift, protein truncation 16, 17, 19 Second allele transcriptionally silent mild CHS compound heterozygote: CGA→TGA, nonsense mutation, codon 50, second 19 mutation not detected late onset CHS homozygous CGA→TGA, nonsense mutation, codon 1103, protein truncation The sequence of the full length LYST protein has interesting features but does not provide any definitive answers as to its function. A 350 amino acid region near to the C-terminus has significant similarities to a number of proteins, including part of another human protein with some similarity to the cell cycle control protein CDC4, but of unknown function, a yeast ORF of unknown function, and three different Caenorhabditis elegans anonymous ORFs. A short 72 amino acid segment of the protein near the N-terminus has limited similarity (26%) to stathmin, which is a protein that regulates microtubule polymerisation. Nagle et al. (17) describe the presence of 23 hydrophobic helices throughout the length of the protein. These are analogous to (but do not have sequence similarity with) repeated motifs called HEAT and ARM which are seen in other proteins. Several proteins with these motifs are involved in vesicle transport, as LYST appears to be. The N-terminal part of Lyst also contains seven repeats of a motif known as WD40, which is thought to be involved in protein/protein interaction. A yeast protein, the product of the VPS15 gene, also has both HEAT/ARM motifs and N-terminal WD40 repeats. VPS15 is one of a large number of genes that are involved in directing proteins vacuole; the yeast lysosome. However, VPS15 is a serine/threonine kinase and is part of a signal transduction mechanism, and there is no evidence for such a function of Lyst. HPS and pale ear HPS (OMIM 203300) is a second human disorder that combines oculocutaneous albinism with a lysosomal storage defect and a bleeding disturbance. Although generally rare, two genetically fairly isolated populations have been noted where it is common; presumably due to a founder effect. Using these populations, from Puerto Rico and from the Swiss Alps, the HPS gene could be mapped to a fairly narrow interval of chromosome 10 (20). Isolation of a YAC and BAC/PAC contig across the region gave rise to more markers that refined the interval to <200 kb using the Puerto Rican patients (21). Two genes were identified in this interval, one of which was mutated in HPS patients. All Puerto Rican patients are homozygous for a 16 bp duplication that results 17 in a frameshift and truncation of the protein (Table 3). All the Swiss individuals are homozygous for a different mutation, due to a single base insertion. This insertion, of an additional C in a run of eight, might be a recurrent mutation as it has been seen in an unrelated Irish patient within a different haplotype. A third mutation has been characterised in a Japanese individual (21) (Table 3). The location of HPS on chromosome 10 permits the identification, by conserved linkage, of the precise region of the mouse genome on chromosome 19 where the mouse HPS homologue must lie. Two closely linked mutations are candidates, ruby eye (ru) and pale ear (ep). Sequencing of the mouse HPS homologue from ru and ep mice found the gene mutated in ep DNA and in a second mutant allele ep 6J (22) (Table 3). The protein encoded by this gene has no obvious features to indicate its function, except for two potential transmembrane domains, suggesting that it is localised in the melanosomal or lysosomal membrane with both termini in the lumen and a cytoplasmic loop (although it has no signal sequence). It bears no similarity to any other proteins in the database, with the exception of a seven or eight amino acid stretch that is identical between LYST and HPS/ep. As both proteins are probably localised in the organellar membrane, this may indicate an interaction with a common protein. However, it is thought that LYST is located on the cytoplasmic side of the membrane, and the peptide motif in HPS/ep is on the lumenal side; the interacting protein would have to span the membrane or be localised on both sides (23). It is interesting to note that all the mutations characterised to date in the human HPS or mouse ep gene result in truncations. Both mouse mutations truncate the protein within 90 amino acids of the C-terminus and two of the three human mutations retain two thirds of the protein, including both transmembrane domains. No differences between the patients have been reported that might correlate with more or less truncated protein products. The mouse ep truncation has lost only 46 amino acid residues (although it also has 92 ‘nonsense’ residues added) and still has a full pale ear phenotype, indicating that the C-terminus of the protein is essential for its function. 1616 Human Molecular Genetics, 1997, Vol. 6, No. 10 Review Table 3. Mutations of the HPS/ep gene Species Mutation Molecular defect Ref. mouse ep IAP insertion, codon 657, protein truncation 22 ep6J 23 bp deletion, 3 bp insertion, codons 611–618, frameshift, protein truncation 22 human Puerto Rican families 16 bp duplication, frameshift codon 496, protein truncation 21 Swiss families single base insertion (duplication), codon 324, frameshift, protein truncation 21 Japanese single base insertion (duplication), codon 441, frameshift, protein truncation 21 The HPS/ep protein may be less important for melanosomal function than for lysosomes and platelets. Whilst pale ear mice have the typical features of lysosome storage diseases and prolonged bleeding time, their hair pigmentation is not severely affected in adults, unlike the pale ear and tail. Pigmentation of ears and tail is produced by melanocytes in the epidermis, whereas hair is pigmented by follicular melanocytes. The density of melanocytes is much greater in the skin and possibly a slight reduction in melanin production is more easily observed in the ear and tail than in the hair. ENDOTHELIN 3 AND MELANOBLAST DEVELOPMENT Melanocytes and neural crest Melanocytes originate during development from the neural crest. These are cells that migrate from either side of the dorsal neural tube soon after it has formed by folding of the neural plate. Neural crest gives rise to numerous cell types, including neuronal and glial cells of the peripheral nervous system, cells of the adrenal gland and craniofacial structures as well as pigment cells. Mutations that affect this process, or a common precursor within the lineages will, of course, affect more than one cell type. The transcription factor PAX3, for example, is required early in the process. Individuals heterozygous for PAX3 mutations have Waardenburg syndrome type 1, and have both abnormal facies and a melanocyte deficiency (24). Splotch mutant mice have mutations in Pax3 and similar defects. When splotch is homozygous it results in lethality due to a severe neural crest defect (25,26). An old classical mouse mutation named piebald (s) specifically affects two neural crest-derived lineages. The mutation is recessive, and when homozygous results in mice that have a variable degree of white spotting. Numerous other alleles of the mutation exist, most of which are more severe. Homozygotes for these mutations lack virtually all pigmentation of the hair and skin (but have dark eyes, as the retinal pigmented epithelium is not of neural crest origin). Furthermore, these animals develop megacolon due to absence of neural crest-derived enteric ganglia and consequently often die. Mice mutant at a second locus, lethal spotting (ls), have a very similar phenotype, with white spots and megacolon. Both mutations are reminiscent of some forms of Hirschsprung disease (HSCR) (OMIM 142623), which is characterised by colonic aganglionosis, and some patients have unpigmented regions, such as a white forelock, eyebrows and eyelashes. About half of familial and 15–20% of sporadic HSCR patients have mutations of the Ret receptor tyrosine kinase (27). Others have deletions on chromosome 13q, and in an inbred pedigree a recessive form of HSCR was mapped to the same region (28). This location is flanked by genes that in the mouse flank the piebald mutation, and thus it has been argued that the two mutant loci are homologous (29). Endothelin receptor-B The identity of the piebald/HSCR gene came from a surprising direction, neither from positional cloning, nor from the expression pattern of a candidate gene. The three endothelin peptides (1, 2 and 3) are recognised by two G protein-coupled heptahelical receptors (A and B). The endothelins were originally identified as vasoconstrictive agents, but when the receptor-B (Ednrb) was mutated via homologous recombination in ES cells, the resulting homozygous mutant mice lacked melanocytes and had megacolon (30). EDNRB maps to chromosome 13 in humans, and 14 in mouse, in the region where piebald/HSCR is located. The ‘knockout’ mutation failed to complement the classical s alleles and the entire Ednrb gene is deleted from piebald-lethal mice. The original s mutation has mRNA expresion reduced by ∼75%. Strictly speaking, none of this genetic data prove that Ednrb is the piebald gene in mice, as there could be an effect on a neighbouring gene, but a mutation in the rat lends convincing evidence. The spotting lethal mutation of rats results in lack of pigmentation and aganglionic megacolon. These animals have a deletion within the Ednrb gene, that results in an aberrantly spliced message and protein truncation (31–33). Initial analysis of EDNRB mutations in the inbred HSCR pedigree showed a complicated inheritance pattern (34). The disease, with or without associated pigmentation defects, was clearly associated with a missense mutation, W276C (Table 4). However, some individuals homozygous for the mutation had no signs of disease, whilst some had only pigmentary defects, indicating reduced penetrance. Furthermore, a few individuals had the disease but were wild-type at the EDNRB locus, indicating additional HSCR genes in the population. Puffenberger et al. (34) suggest that modifier genes in the population will affect penetrance, and point to RET as one such modifier Another candidate modifier would be the ligand of RET, GDNF, and recent data suggest that variants of this gene do modify the penetrance of RET-mutant HSCR (35,36). In mice, the expressivity of the s mutation is clearly under genetic control. Two different strains of mice with the same mutation have a substantial difference in the extent of white spotting. A backcross showed that these differences were controlled by genetic background. The major locus influencing expressivity maps at or near the mast cell growth factor (Mgf) gene, which is the gene affected by spotting mutations called steel, and which encodes the ligand for the melanocyte survival factor, Kit (37). 1617 Human Molecular Genetics, 1997,1994, Vol. Vol. 6, No. Review 1617 Nucleic Acids Research, 22,10 No. 1 Table 4. Mutations of the endothelin receptor B gene Species Mutation Molecular nature mouse s 4× decrease in mRNA; molecular nature not defined 30 rat sl sl complete gene deletion 301 bp deletion: abnormal splicing; two mRNAs, one with frameshift, protein truncation, one with in frame internal deletion heterozygous with wild-type gene; large chromosomal deletion; gene deletion heterozygous with wild-type gene; 1 bp insertion codon 293, frameshift, protein truncation heterozygous with wild-type gene; 1 bp deletion codon 378, frameshift, protein truncation heterozygous with wild-type gene; codon 275, TGG→TAG, nonsense mutation, protein truncation heterozygous with wild-type gene; codon 57, GGT→AGT, Gly→Ser heterozygous with wild-type gene; codon 305, AGT→AAT, Ser→Asn heterozygous with wild-type gene; codon 319, GGG→TGG, Arg→Trp heterozygous with wild-type gene; codon 383, CCA→CTA, Pro→Leu heterozygous with wild-type or homozygous; codon 276, TGG→TGT, Trp→Cys 30 31–33 115 116 117 116 115 117 115 115 34 homozygous; codon 183, GCC→GGC, Ala→Gly 118 heterozygous with wild-type gene; G→A in 5′ UTR. Causation not proven 115 human HSCR HSCR HSCR (partially penetrant)a HSCR HSCR (partially penetrant)a HSCR (partially penetrant)a HSCR (partially penetrant)a HSCR (partially penetrant)a HSCR (partially penetrant)a; sometimes pigmentation defect Waardenburg–Hirschsprung disease (Shah–Waardenburg) HSCR (partially penetrant)a aPenetrance Ref. of others not necessarily determined. Further analysis of the EDNRB gene in HSCR patients may have clarified the inheritance patterns. Most patients are heterozygous for a mutant allele. Two sisters have been described who are homozygous for a missense mutation, A183G (Table 4). These girls both had pigmentary defects, including a white forelock, heterochromia irides and deafness (due to lack of inner ear melanocytes) as well as colonic aganglionosis. Their parents and a carrier brother were symptom free. However, at least 12 cases have now been found in which individuals with short segment colonic aganglionosis are heterozygous for a deletion, missense, nonsense or frameshift mutation of EDNRB (Table 4). None of these cases show any pigmentary defect, and all have inherited the mutation from parents who have no sign of HSCR. Presumably genetic background is playing a role in penetrance of the disease. It should be borne in mind, however, that there has been a case of monozygotic twins, one of whom has HSCR and one who does not. There must also be stochastic events during development in addition to genetic influences that affect the colonic ganglion population. Endothelin 3 At the same time as the targeted mutation in mice of the Ednrb mutation was reported, a knockout of the gene encoding one of the ligands of this receptor, endothelin-3 (Edn3) was also described. Mice homozygous for this mutation also have a white spotting phenotype and aganglionic megacolon. The white spotting is less severe than results from a loss of Ednrb; melanocytes remain on the head and hips. The Edn3 gene maps on distal chromosome 2, where a mutation with a similar phenotype, lethal spotting (ls) lies. Active endothelins are processed from large prepropeptides. These are cleaved to inactive intermediates, big endothelins, which are further acted on by a specific converting enzyme to produce the 21-residue active peptide. The Edn3 gene in ls mice contains a missense mutation within the sequence cleaved to convert the prepropeptide to big endothelin (Table 5), which blocks formation of the mature peptide. Two patients have subsequently been found who are homozygous for mutations in the human EDN3 gene (Table 5). Both have total absence of enteric ganglia in the gut, and both have pigmentary defects including a white forelock, white eyelashes, white skin patches and deafness. The parents of both patients had no symptoms, but several relatives in one family (one of whom was demonstrated to be a carrier) had a range of pigmentation defects, but did not have HSCR. Another patient has been found with an apparent EDN3 mutation who has neither HSCR nor pigmentary anomalies. Instead they have congenital central hypoventilation syndrome (CCHS), a disease which causes patients to hypoventilate when asleep. CCHS is often associated with HSCR, but may occur alone. In this case the mutation, a frameshift truncating the last 41 amino acids of the precursor protein does not appear to be a polymorphism (it is not found in 260 control chromosomes). However, it does not affect processing of the prepropeptide in a cultured cell assay and definitive proof that this mutation causes CCHS awaits further work. It is worth noting the differences between mouse and human inheritance of these mutations. Mice heterozyogus for a loss of function of either the receptor or the ligand have normal ganglia throughout their gut (38), whilst human patients carrying one clear loss of function mutation of the receptor have short segment colonic aganglionosis, but no pigmentary defects. (Assuming that the EDNRB allele assessed as normal does not have, for example, reduced expression.) Patients with a mutation in both EDNRB alleles have a variable extent of pigmentary defects in association with aganglionosis; but neither mutation found as homozygous to date is definitely a complete loss of function. In fact the fairly mild pigmentary anomalies might suggest some residual receptor activity is present. The patients homozygous for a clear loss of function of EDN3 have a very severe aganglionosis and pigmentation defects, although the unpigmented patches seems to be less extensive than those seen in lethal-spotted mice. 1618 Human Molecular Genetics, 1997, Vol. 6, No. 10 Review Table 5. Mutations of the endothelin-3 (EDN3/Edn3) gene Species Mutation Molecular defect Ref. mouse lethal spotting, ls codon 137, CGG→TGG, Arg→Trp; abolish peptide processing 119 human Waardenburg–Hirschsprung disease (Shah-Waardenburg) homozygous; 2 bp deletion, 1 bp insertion, codon 88; frameshift, protein truncation 120 Waardenburg-Hirschsprung disease (Shah-Waardenburg) homozygousa; 121 congenital central hypoventilation syndrome heterozygous with wild-type gene; 1 bp insertion codon 188, frameshift, termination aHeterozygous codon 159, TGC→TTC, Cys→Phe 122 relatives have partially penetrant pigmentary defects. Role of Ednrb/Edn3 interaction Many questions are raised as to the role of this receptor/ligand interaction in the development of melanocytes and enteric neurons. Mutations that affect both cell types could be acting in two ways. Either the interaction is needed in two lineages, both of neural crest origin, but independently in the lineages after separation, or the interaction might be needed for the development, proliferation or survival of a progenitor of both lineages. Using an in situ probe for the melanoblast lineage, Pavan and Tilghman (39) showed that no positive cells could be detected emerging from the neural crest of Ednrb s–l homozygotes, save for a few near the tail where pigmented patches were often seen. Two groups have shown that endothelin-3 in culture acts synergistically with other factors to increase the number of melanocyte progenitors (40,41). The interaction is clearly needed early in melanoblast development, but whether it is needed before the enteric ganglia and melanoblast lineage diverge is not known. Why some cells escape the requirement for Edn3 is not known. Edn3 acts in culture to promote differentiation to a pigmented phenotype and hence can also act late in development (40). There is a temporal difference in emergence of the cells from the crest. In general neuronal progenitor cells leave the crest early, whilst melanoblasts leave late (42). Kapur et al. (38) used a transgenic lineage marker to track the migration into the gut by crest-derived neuroblasts in normal and Ednrb s–l mouse embryos. Colonisation of the gut occurs in a proximal→distal (cranial→caudal) direction. Neuroblasts appeared normally in the proximal gut early in development of mutant embryos but at embryonic day 12.5 there was a transient arrest, followed by slower migration to the distal parts of the gut, parts of which were never colonised. It seems that it is the later colonising cells that are affected by loss of the endothelin receptor. It may be that, for some reason, only later emerging cells require the receptor–ligand interaction to survive or proliferate (and the melanoblasts, which also emerge late also need it). Alternatively, all enteric neuroblasts may be affected and have reduced proliferation or survival, and the diminished number or viability of precursors is insufficient to affect small intestinal colonisation, but does affect colonisation of the colon. Very similar results have been obtained with Edn3 mutant embryos. Kapur et al. (38) also made a very surprising discovery using chimaeras between wild-type embryos and Ednrb s–l mutant, transgene-tagged embryos. Homozygous mutant enteric ganglion cells, which normally never reach the colon, can be rescued by wild-type cells, so that in chimaeras they can be seen throughout the length of the gut. These rescued cells have a deletion of the receptor gene, and yet must receive a signal from nearby cells that promotes their development. There are also data to indicate that Edn3 is produced by the enteric ganglion cells themselves and acts in an autocrine or paracrine fashion. It seems likely that in response to Edn3, via the Ednrb, neighbouring cells produce another intercellular signal which, in turn, acts back on the ganglion cells. So Ednrb mutant cells could still trigger this signal from neighbouring cells, and still respond to develop normally. The nature of this signal is unknown, but one candidate might be glial-derived neurotrophic factor, GDNF, the ligand for RET, which is, of course, the other receptor shown genetically to be required for enteric ganglia colonisation of the gut. Mayer (43) has also demonstrated, by coculturing mutant neural tube with wild-type or mutant skin, that Ednrb mutant melanocytes survive better in wild-type skin. The mutation therefore is not melanoblast autonomous. A similar intercellular signalling process downstream of Ednrb may operate on melanoblasts as in enteric ganglia. A candidate for this signal may be Mgf, the ligand of the Kit receptor. Reid et al. (40) show that Edn3 and Mgf act synergistically on neural tube cultures to increase the number of melanocyte progenitors by >10-fold over the effect of either alone. MELANOCYTE-STIMULATING HORMONE RECEPTOR AND αMSH Melanocytes can synthesise two different types of melanin: the black or brown eumelanin and the yellow or red phaeomelanin. Genetic evidence from several species indicates that the type of melanin made is determined by response to a family of peptide hormones known as melanocortins, mediated at the melanocyte by a G-protein coupled, heptahelical receptor, the melanocortin-1 receptor (Mc1r) (44,45). Four peptides which derive from the precursor protein, pro-opiomelanocortin (POMC) have melanocortin activity. In culture, melanocytes respond to melanocortins by altering their morphology, increasing expression of a number of melanogenic enzymes and increasing the activity of tyrosinase enzyme which leads to an increase in melanin synthesis (46–48). The cells respond to the peptides differentially, with αMSH being the most potent, at least in mouse cells, although ACTH, a larger peptide that includes αMSH at its N-terminus may be more active on human melanocytes (49). POMC is synthesised in the pituitary from where circulating melanocortins derive, and melanocortins in the blood can affect epidermal or follicular melanocytes. Increased levels of circulating ACTH, as in Addison’s disease in humans, causes hyperpigmentation, and the increased plasma αMSH in dopamine D2 receptor mutant mice (50) increases eumelanin synthesis. However, the pituitary-derived melanocortins may not be the peptides that act on melanocytes under normal conditions, as animals from which the pituitary has been removed are normally pigmented. It is likely that POMC 1619 Human Molecular Genetics, 1997,1994, Vol. Vol. 6, No. Review 1619 Nucleic Acids Research, 22,10 No. 1 Figure 1. Amino acid sequence alignment of MC1R from six species (the horse sequence is incomplete). Transmembrane domains 1–7 are indicated by overlining. Numbering is according to the human sequence. Variant amino acid residues are in bold and underlined. The alterations are as follows. Mouse: S69L, Mc1r tob (dominant black); E92K, Mc1r so–6J (dominant black); l98P, Mc1r so (dominant black); deletion at codon 183, frameshift (recessive yellow) (53). Cow: L99P, dominant black; deletion at codon104, frameshift, recessive red (54,55). Horse: S83F, chestnut (56). Fox: C125R, silver (57). Chicken: D92K (plus M92T, A143T, R213C) E (extended black); C33W (plus D37G) e y (recessive yellow) (58). (Note the chicken variants each occur on a different haplotype background.) Human: V60L; A64S; F76Y; D84E; V92M; T95M; V97I; A103V; L106Q; R151C; I155T; R160W; R163Q; D294H (59; E.Healy and J.Rees, personal communication). synthesised locally by keratinocytes is the physiological source of melanocortins that act on melanocytes in the epidermis and hair follicle (51). Mutations of melanocortin receptor-1 There is a family of five melanocortin receptors (44,52), one of which, Mc1r, is expressed specifically in melanocytes. The hair of mice is pigmented by both eumelanin and phaeomelanin, the distribution of which is determined by activity of Mc1r, which in turn is normally modulated by the action of agouti protein (see below). A loss of function mutation of Mc1r in mice, recessive yellow (e) (Fig. 1) results in animals which produce only phaeomelanin in the follicular melanocytes, and are completely yellow. Three other mutant alleles in the same gene have the opposite phenotype; the mice produce predominantly eumelanin. These dominant mutations result in mice that have constitutive activation of Mc1r or that are hyper-responsive to melanocortins (Fig. 1), and all are missense mutations within the second transmembrane domain of the protein (53). Recently mutations in MC1R have been found in a number of other species, although where amino acid substitutions are found, analysis of the pharmacological function of the changes has not usually been carried out. In a few cases the mutation is clearly a loss of function. Red guinea pigs have a deletion of the gene (45) 1620 Human Molecular Genetics, 1997, Vol. 6, No. 10 Review and red cattle have a frameshift in MC1R, which must inactivate the protein (54,55) (Fig. 1). The same base change is found in red Norwegian Cattle and red Holsteins, indicating a common origin for the mutation in domestic cattle. There is a black variant of Norwegian Cattle, and the MC1R gene in these animals has a missense mutation in the second transmembrane domain very similar to the constitutively active dominant mouse Mc1r variant (54) (Fig. 1). The chestnut phenotype of horses cosegregates with a substitution of a conserved amino acid residue in the second transmembrane domain of MC1R, which is likely to inactivate the receptor (56). Surprisingly, the coat colour of the red fox is not due to a variant of MC1R. However, a semi-dominant variant of the gene, which produces a constitutively active protein, appears to be responsible for darkening of the coat of certain fox strains (57) (Fig. 1). There is an interaction between this allele and a variant at the agouti locus which modifies the heterozygous phenotype, but homozygosity for the dominant variant invariably results in the so-called silver fox phenotype. Variants in the gene also affect feather pigmentation in chickens. Chicken with a dominant mutation that gives rise to uniformly black feathers have a mutation on the second transmembrane domain that results in an amino acid substitution identical to one of the dominant black alleles of mouse (58) (Fig. 1), although several other additional changes are seen throughout the protein. The recessive variant that produces a uniform yellow/red feather pigmentation is associated with three missense mutations, two of which are in codons that are completely conserved throughout all six published MC1R sequences (58) (Fig. 1). It is likely, though remains to be proven, that one or both of these mutations cause loss of MC1R function. Human variation in MC1R The involvement of this gene in hair pigmentation variation in so many species prompted several groups to look for an association of variants of the human gene with red hair colour. In humans skin pigmentation shows clear variation that is partially independent of hair colour. Skin pigmentation in the caucasian population can be classified into four groups according to the degree of sensitivity to sunlight; ranging from type I skin which always burns and never tans, to skin type IV which tans well and rarely burns. All skin types can be found in association with all hair colours from blond and fair to dark brown or black, apart from red hair which is invariably only seen in individuals with skin type I or II. Valverde et al. (59) found a clear association between red haired individuals and variants in the MC1R gene (Fig. 1). As all redheads have the lighter skin types, then the variants could be producing pale skin rather than red hair and this appears to be the case. Variants have been found in subjects who have fair or dark hair and skin type I or II (59–61; E. Healy and J. Rees, personal communication). In the absence of family studies the genetics are not as yet clear. Redhaired/pale skinned individuals may be homozygous or heterozygous for MC1R variants, or possibly have no variant at all (although it is difficult to prove that a gene with wild-type sequence is not expressed at a lower level than normal, for example). There does not seem to be any phenotypic difference between homozygotes and heterozygotes, and there may well be interactions with other genes. Barsh (62) has expressed the data as a ‘relative risk’ for having red hair, being 15-fold increased when heterozygous and 170-fold when homozygous. It is possible that variation in the gene in humans results in pale skin, which is a necessary condition for red hair, which is thus due to the action of other genes whose activity is only seen on an MC1R variant background. Perhaps it is significant that not one of the numerous variants is a frameshift or nonsense mutation which would be a convincing null. All are missense mutations whose functional consequences have yet to be determined. Many of the amino acid changes described thus far are in the second transmembrane domain, where numerous variants have been seen in other species, albeit in these cases producing a dominant, darkening phenotype. Some of the human variants are in positions that are not well conserved between species, and thus their consequences are difficult to predict. Other variants are conservative substitutions or indeed may even introduce an amino acid seen in that location in other species, and these may be neutral polymorphisms in the human population. Nevertheless, some changes would appear on the basis of sequence comparison between species to be functionally significant, but the key test is to assay the function of the variants. Two variants have been tested by expression in heterologous cells and measuring the rise in intracellular cAMP in response to αMSH, and neither of these (Val92Met and Asp84Glu) show significant variation from wild-type response (60). The Val92Met variant has also been assayed for binding affinity by αMSH, and it appears to have a 5-fold lower affinity for the hormone than wild-type (63). A more physiological and sensitive assay will be to use transgenic mice to assay the ability of these variants to rescue the loss of function Mc1re mutation. A major risk factor for all forms of skin cancer is the number of sunburn episodes. Having a paler skin type by definition increases the chance of sunburn, and thus these skin types confer an increased risk of cancer. One study has found a highly significant association between MC1R variants and melanoma (64). In fact the genetic variants were better predictors of melanoma risk than skin type. Two explanations might account for this. Firstly, determination of skin type is a crude measurement, and this study in some cases used telephone or third party information to assign skin type. Assay of genetic variation might then be a more accurate and objective method of assigning skin type. Alternatively, there may be more than one genetic cause of pale skin, not all of which increase the risk of melanoma equally, and MC1R variants may disproportionately increase the risk. In either case the MC1R genotype may be a useful measure of an individual’s melanoma risk. Agouti: an antagonist of melanocortin signalling One mouse gene for which the genetics is very well understood, but for which no human variants have been described, is the agouti gene, which encodes a polypeptide antagonist of Mc1r. Each mouse dorsal hair is patterned by a band of phaeomelanin flanked by bands of eumelain which is produced by the expression of a pulse of agouti protein in the skin midway through the growth of the hair. The presence of agouti protein blocks the action of αMSH, and switches melanogenesis from production of eumelanin (which is dependent on αMSH) to phaeomelanin. The agouti gene in dorsal skin has a temporally regulated promoter, synchronised with the hair growth cycle (65). Wild-type, white bellied agouti, mice have a phaeomelanic ventrum, in which the agouti gene is constantly expressed through use of a different, spatially regulated, promoter (66). 1621 Human Molecular Genetics, 1997,1994, Vol. Vol. 6, No. Review 1621 Nucleic Acids Research, 22,10 No. 1 Recessive, loss of function mutations of agouti produce mice that are uniformly black. One of the old mouse mutations, nonagouti (a) has a coat that is almost entirely black, but still has a few yellow hairs. In these mice the agouti gene is not entirely inactivated, but has a double, or nested, insertion of retroposons upstream, which inactivates both spatially and temporally regulated promoters throughout most of the skin, but still allows expression in certain locations. The a allele can revert by excision of the retroposons via recombination of the LTRs. If the ‘inner’ retroposon is excised, which leaves behind the outer one, the ventral specific promoter is restored, but the temporally regulated promoter is not, giving rise to animals that have a phaeomelanic belly but a black back. If both retroposons are excised, then both promoters are restored (67). Loss of function of the agouti gene can explain recessive black phenotypes in a number of species, including dogs and cattle, but molecular data is only available for foxes (57). A deletion of exon 2 of the fox agouti gene produces a semi-dominant phenotype that darkens the legs and tail, but leaves the rest of the body phaeomelanic. Homozygous animals have the same silver coat that homozygotes for the MC1R produce. The agouti and MC1R genes clearly interact; the double heterozygote is darker than either single heterozygote, but less dark than either homozygote. Gain of function, dominant mutations of mouse agouti are extremely interesting, and are the reason why agouti protein has attracted a lot of attention recently. Numerous different mouse alleles exist, but all have promoter rearrangements, deletions or insertions that result in mice that have misregulation of agouti protein expression (68). Expression in the skin is permanently on, so the Mc1r is permanently blocked and the melanocytes make only phaeomelanin. Expression in one or more other locations has the result that the animals become obese, presumably because the protein interferes with some mechanism of weight or feeding homeostasis (see below). The mechanism of agouti antagonism in Mc1r signalling is not understood in detail. Recombinant agouti protein is an antagonist of the melanocortin induced increase in intracellular cAMP in heterologous cells that express mouse Mc1r or in melanoma cells and will prevent the enhanced synthesis of melanin by melanoma cells in response to αMSH (69–71). There has been debate as to whether agouti acts to block melanocortin at the receptor or whether it binds to a different receptor and produces antagonism intracellularly. The evidence is now strong that action on pigment cells is through Mc1r. Agouti protein can be shown directly to bind to the receptor (69,71). On the other hand, agouti protein can elicit a response in melanoma cells that is independent of αMSH. Cells treated with the recombinant protein have dose-dependent growth inhibition, clearly showing an effect of agouti that is not due to antagonism of Mc1r. However, this activity is also mediated through Mc1r, as melanoma cells that lack the receptor do not show the response (71). The likeliest explanation is that agouti is an inverse agonist of Mc1r; that is, it can compete with αMSH for binding to the receptor and on binding acts to reduce the basal (unstimulated) signalling from Mc1r. Recessive yellow mice (Mc1r deficient) are not identical to dominant yellow animals (over-expressing agouti); they are a darker shade. This is consistent with agouti having an effect on melanocytes that is more than simply blocking αMSH. A genetic prediction which ensues is that a double mutant, both lacking Mc1r and overexpressing agouti will have a phenotype the same as the recessive yellow animals, because the overexpressed agouti protein cannot act on Mc1r deficient melanocytes. Agouti protein and obesity Numerous different, dominant overexpressing, alleles of the mouse agouti gene all demonstrate obesity (65,68). Furthermore, transgenic mice, in which the agouti gene is expressed from a ubiquitous promoter are also obese (72). The simplest model to explain this is that agouti protein is antagonising another melanocortin receptor, which alters the regulation of weight control or feeding. A corollary is that there might be other proteins, related to agouti, that are expressed in the brain that are the normal antagonist of these receptors. Agouti has been shown to antagonise other members of the melanocortin receptor family. Mc3r and Mc5r are not affected by concentrations of agouti protein that antagonise Mc1r. However agouti is a potent antagonist of the brain-expressed receptor Mc4r (69). The role of Mc4r in regulating mouse feeding behaviour has been elegantly demonstrated by both genetic and pharmacological means. An ES-cell mediated gene disruption of Mc4r, when homozygous, results in mice that are hyperphagic and obese (73). The synthetic cyclic melanocortin compound SHU9119 is a potent antagonist of αMSH binding to Mc4r, but not to Mc3r. When normal mice are injected in the cerebral ventricles with SHU9119 they respond by increasing food intake (74). By contrast, a very closely related compound, MTII has the reverse effect, being a potent agonist of Mc4r, but less so of Mc3r. When MTII is injected intracerebrally, feeding is inhibited (74). If modulating Mc4r activity by artificial means can have these effects on feeding, is there a natural neuropeptide that has the same effect? By screening the sequence database Shutter et al. (75) identified a protein distantly related to agouti (ART), that is expressed in the mouse brain in a similar location to POMC and to neuropeptide Y, another peptide hormone that can influence feeding behaviour. ART may be the natural antagonist of Mc4r and may be involved in regulation of feeding and weight. Coming to understand the nature of a number of classical mouse mutations has led to potentially very important insights into the regulation of feeding behaviour. 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